Six Month Exercise

A Six Months Exercise Intervention Influences the
Genome-wide DNA Methylation Pattern in Human

Adipose Tissue
Tina Ronn1*, Petr Volkov1, Cajsa Davegardh1, Tasnim Dayeh1, Elin Hall1, Anders H. Olsson1,
sa Tornberg2, Marloes Dekker Nitert3, Karl-Fredrik Eriksson4, Helena A. Jones5,
Emma Nilsson1, A
6
Leif Groop , Charlotte Ling1*
1 Department of Clinical Sciences, Epigenetics and Diabetes, Lund University Diabetes Centre, CRC, Malmo, Sweden, 2 Department of Health Sciences, Division of Physiotherapy,
Lund University, Lund, Sweden, 3 School of Medicine, Royal Brisbane Clinical School, The University of Queensland, Herston, Queensland, Australia, 4 Department of Clinical
Sciences, Vascular Diseases, Lund University, Malmo, Sweden, 5 Department of Experimental Medical Science, Division of Diabetes, Metabolism and Endocrinology, Lund
University, BMC C11, Lund, Sweden, 6 Department of Clinical Sciences, Diabetes and Endocrinology, Lund University Diabetes Centre, CRC, Malmo, Sweden
Abstract
Epigenetic mechanisms are implicated in gene regulation and the development of different diseases. The epigenome differs
between cell types and has until now only been characterized for a few human tissues. Environmental factors potentially
alter the epigenome. Here we describe the genome-wide pattern of DNA methylation in human adipose tissue from 23
healthy men, with a previous low level of physical activity, before and after a six months exercise intervention. We also
investigate the differences in adipose tissue DNA methylation between 31 individuals with or without a family history of
type 2 diabetes. DNA methylation was analyzed using Infinium HumanMethylation450 BeadChip, an array containing
485,577 probes covering 99% RefSeq genes. Global DNA methylation changed and 17,975 individual CpG sites in 7,663
unique genes showed altered levels of DNA methylation after the exercise intervention (q,0.05). Differential mRNA
expression was present in 1/3 of gene regions with altered DNA methylation, including RALBP1, HDAC4 and NCOR2 (q,0.05).
Using a luciferase assay, we could show that increased DNA methylation in vitro of the RALBP1 promoter suppressed the
transcriptional activity (p = 0.03). Moreover, 18 obesity and 21 type 2 diabetes candidate genes had CpG sites with
differences in adipose tissue DNA methylation in response to exercise (q,0.05), including TCF7L2 (6 CpG sites) and KCNQ1
(10 CpG sites). A simultaneous change in mRNA expression was seen for 6 of those genes. To understand if genes that
exhibit differential DNA methylation and mRNA expression in human adipose tissue in vivo affect adipocyte metabolism, we
silenced Hdac4 and Ncor2 respectively in 3T3-L1 adipocytes, which resulted in increased lipogenesis both in the basal and
insulin stimulated state. In conclusion, exercise induces genome-wide changes in DNA methylation in human adipose tissue,
potentially affecting adipocyte metabolism.
Citation: Ronn T, Volkov P, Davegardh C, Dayeh T, Hall E, et al. (2013) A Six Months Exercise Intervention Influences the Genome-wide DNA Methylation Pattern
in Human Adipose Tissue. PLoS Genet 9(6): e1003572. doi:10.1371/journal.pgen.1003572
Editor: John M. Greally, Albert Einstein College of Medicine, United States of America
Received January 4, 2013; Accepted May 2, 2013; Published June 27, 2013
Copyright: 2013 Ronn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Swedish Research Council (CL and LG) and Lund University Diabetes Centre (LUDC), the Knut & Alice
Wallenbergs stiftelse, Fredrik & Ingrid Thurings stiftelse (TR), Kungliga Fysiografiska sallskapet (TR), Tore Nilssons stiftelse (TR), Pahlssons stiftelse (CL), Novonordisk
foundation (CL), ALF (CL), Diabetes forbundet (CL), Soderbergs stiftelse (CL) and by an EU grant (ENGAGE; LG). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: tina.ronn@med.lu.se (TR); charlotte.ling@med.lu.se (CL)
the disease [46]. Individuals with high risk of developing T2D

strongly benefit from non-pharmacological interventions, involving diet and exercise [7,8]. Exercise is important for physical
health, including weight maintenance and its beneficial effects on
triglycerides, cholesterol and blood pressure, suggestively by
activating a complex program of transcriptional changes in target
tissues.
Epigenetic mechanisms such as DNA methylation are considered to be important in phenotype transmission and the
development of different diseases [9]. The epigenetic pattern is
mainly established early in life and thereafter maintained in
differentiated cells, but age-dependent alterations still have the
potential to modulate gene expression and translate environmental
factors into phenotypic traits [1013]. In differentiated mamma-
Introduction
A sedentary lifestyle, a poor diet and new technologies that
reduce physical activity cause health problems worldwide, as
reduced energy expenditure together with increased energy intake
lead to weight gain and increased cardiometabolic health risks [1].
Obesity is an important predictor for the development of both type
2 diabetes (T2D) and cardiovascular diseases, which suggests a
central role for adipose tissue in the development of these
conditions [2]. Adipose tissue is an endocrine organ affecting
many metabolic pathways, contributing to total glucose homeostasis [2]. T2D is caused by a complex interplay of genetic and
lifestyle factors [3], and a family history of T2D has been
associated with reduced physical fitness and an increased risk of
PLOS Genetics | www.plosgenetics.org
June 2013 | Volume 9 | Issue 6 | e1003572
Exercise and Human Adipose Tissue DNA Methylation
476,753 sites. No individual CpG site showed a significant

difference in DNA methylation between FH+ and FH2 men after
false discovery rate (FDR) correction (q.0.05) [24]. Additionally,
there were no global differences between the FH+ and FH2
individuals when calculating the average DNA methylation based
on genomic regions (Figure 1a) or CpG content (Figure 1b;
q.0.05).
Author Summary
Given the important role of epigenetics in gene regulation
and disease development, we here present the genomewide DNA methylation pattern of 476,753 CpG sites in
adipose tissue obtained from healthy men. Since environmental factors potentially change metabolism through
epigenetic modifications, we examined if a six months
exercise intervention alters the DNA methylation pattern
as well as gene expression in human adipose tissue. Our
results show that global DNA methylation changes and
17,975 individual CpG sites alter the levels of DNA
methylation in response to exercise. We also found
differential DNA methylation of 39 candidate genes for
obesity and type 2 diabetes in human adipose tissue after
exercise. Additionally, we provide functional proof that
genes, which exhibit both differential DNA methylation
and gene expression in human adipose tissue in response
to exercise, influence adipocyte metabolism. Together, this
study provides the first detailed map of the genome-wide
DNA methylation pattern in human adipose tissue and
links exercise to altered adipose tissue DNA methylation,
potentially affecting adipocyte metabolism.
Clinical outcome and global changes in adipose tissue

DNA methylation in response to exercise
Subcutaneous adipose tissue biopsies were taken from 23 men
both before and after exercise, followed by successful DNA
extraction and analysis of DNA methylation using the Infinium
HumanMethylation450 BeadChip array. Since we found no
significant differences in DNA methylation between FH+ and
FH2 men at baseline, the two groups were combined when
examining the impact of exercise on DNA methylation in adipose
tissue. In Table 1 the clinical and metabolic outcomes of the
exercise intervention are presented for these 23 men, showing a
significant decrease in waist circumference, waist/hip ratio,
diastolic blood pressure, and resting heart rate, whereas a
significant increase was seen for VO2max and HDL.
To evaluate the global human methylome in adipose tissue, we
first calculated the average level of DNA methylation in groups
based on either the functional genome distribution (Figure 1a), or
the CpG content and neighbourhood context (Figure 1b). We also
present the average level of DNA methylation separately for the
Infinium I (n = 126,804) and Infinium II (n = 326,640) assays due
to different b-value distributions for these assays [25]. When
evaluating Infinium I assays in relation to nearest gene, the global
level of DNA methylation after exercise increased in the 39
untranslated region (UTR; q,0.05), whereas a decrease was seen
in the region 1500200 bp upstream of transcription start
(TSS1500), TSS200, 59UTR and within the first exon (1st Exon;
q,0.05). The global DNA methylation level of Infinium II assays
increased significantly (q,0.05) after exercise within all regions
except TSS200 (Figure 1c and Table S2). In general, the average
level of DNA methylation was low in the region from TSS1500 to
the 1st Exon (536%), whereas the gene body, the 39UTR and
intergenic region displayed average DNA methylation levels
ranging from 4372% (Figure 1c and Table S2). When evaluating
global DNA methylation based on CpG content and distance to
CpG islands, average DNA methylation for Infinium I assays
decreased significantly after exercise in CpG islands, whereas an
increase was seen in northern and southern shelves (regions 2000
4000 bp distant from CpG islands) as well as in the open sea
(regions further away from a CpG island) (q,0.05; Figure 1d and
Table S2). For Infinium II assays, average DNA methylation was
significantly increased in all regions after the exercise intervention
(q,0.05; Figure 1d and Table S2). The global level of DNA
methylation was low within CpG islands (921%), intermediate
within the shores (2000 bp regions flanking the CpG islands; 31
44%), whereas the shelves and the open sea showed the highest
level of DNA methylation (6776%; Figure 1d and Table S2).
Although technical variation between probe types has been
reported for the Infinium HumanMethylation450 BeadChip
array, seen as a divergence between the b-values distribution
retrieved from the Infinium I and II assays [25], the global
differences in DNA methylation we observe between probe types
are more likely a result of skewed GC content due to the design
criteria of the two different assays. Infinium I assays have
significantly more CpGs within the probe body than the Infinium
II assays, and 57% are annotated to CpG islands, whereas most
lian cells, DNA methylation usually occurs in the context of CG

dinucleotides (CpGs) and is associated with gene repression [14].
Changes in epigenetic profiles are more common than genetic
mutations and may occur in response to environmental, behavioural, psychological and pathological stimuli [15]. Furthermore,
genetic variation not associated with a phenotype could nonetheless affect the extent of variability of that phenotype through
epigenetic mechanisms, such as DNA methylation. It is not known
whether epigenetic modifications contribute to the cause or
transmission of T2D between generations. Recent studies in
human skeletal muscle and pancreatic islets point towards the
involvement of epigenetic modifications in the regulation of genes
important for glucose metabolism and the pathogenesis of T2D
[11,12,1621]. However, there is limited information about the
regulation of the epigenome in human adipose tissue [22].
The mechanisms behind the long-lasting effects of regular
exercise are not fully understood, and most studies have focused on
cellular and molecular changes in skeletal muscle. Recently, a
global study of DNA methylation in human skeletal muscle
showed changes in the epigenetic pattern in response to long-term
exercise [23]. The aims of this study were to: 1) explore genomewide levels of DNA methylation before and after a six months
exercise intervention in adipose tissue from healthy, but previously
sedentary men; 2) investigate the differences in adipose tissue DNA
methylation between individuals with or without a family history
of T2D; 3) relate changes in DNA methylation to adipose tissue
mRNA expression and metabolic phenotypes in vitro.
Results
Baseline characteristics of individuals with (FH+) or
without (FH2) a family history of type 2 diabetes
A total of 31 men, 15 FH+ and 16 FH2, had subcutaneous
adipose tissue biopsies taken at baseline. The FH+ and FH2
individuals were group-wise matched for age, gender, BMI and
VO2max at inclusion, and there were no significant differences
between FH+ and FH2 individuals, respectively (Table S1). DNA
methylation in the adipose tissue was analyzed using the Infinium
HumanMethylation450 BeadChip array. After quality control
(QC), DNA methylation data was obtained for a total number of
Figure 1. Location of analyzed CpG sites and global DNA methylation in human adipose tissue. All CpG sites analyzed on the Infinium
HumanMethylation450 BeadChip are mapped to gene regions based on functional genome distribution (A) and to CpG island regions based on CpG
content and neighbourhood context (B). In the lower panels, global DNA methylation in human adipose tissue is shown for each gene region (C) and for
CpG island regions (D). Global DNA methylation is calculated as average DNA methylation based on all CpG sites in each region on the chip, and presented
separately for Infinium I and Infinium II assays, respectively. Data is presented as mean 6 SD. TSS, proximal promoter, defined as 200 bp (basepairs) or
1500 bp upstream of the transcription start site; UTR, untranslated region; CpG island, 200 bp (or more) stretch of DNA with a C+G content of 50% and an
observed CpG/expected CpG in excess of 0.6; Shelf, regions flanking island shores, i.e., covering 20004000 bp distant from the CpG island; Shore: the
flanking region of CpG islands, 02000 bp. *Significant difference between average DNA methylation before versus after exercise, q,0.05.
doi:10.1371/journal.pgen.1003572.g001
Infinium II assays have less than three underlying CpGs in the

probe and only 21% are designated as CpG islands [26].
[27] showed only one probe with 50 bases and 14 probes with 49
bases matching to an alternative genomic location. Data of the
most significant CpG sites (q,0.005) and the sites that exhibit the
greatest change in adipose tissue DNA methylation (difference in
DNA methylation .8%) in response to exercise are presented in
Table 23 and included ITPR2 and TSTD1 for increased, and
LTBP4 for decreased DNA methylation. We found 7 CpG sites in
this list to be targeted by Infinium probes reported to cross-react to
alternative genomic locations (47 or 48 bases) [27]. Additionally, to
investigate the possibility that the changes we see in response to
exercise is rather an effect of epigenetic drift over time, we
compared our 1,009 differentially methylated CpG sites (q,0.05,
difference in b-value.5%) with three studies reporting agingdifferentially methylated regions (a-DMRs) in a total of 597 unique
positions [2830]. Secondly we tested for association between age
and the level of DNA methylation in the 31 individuals included at
baseline in this study, representing a more valid age range (3045
years) and tissue for the current hypothesis. We found no overlap
between previously published a-DMRs or the age-associated CpG
sites within our study (18 CpG sites; p,161025), and the CpG
sites differentially methylated after the exercise intervention.
The genomic distribution of individual CpG sites with a
significant change in DNA methylation $5% with exercise is
shown in Figure 3cd, in comparison to all probes located on the
Infinium HumanMethylation450 BeadChip and passing QC. The
distribution is based on location in relation to the functional genome
distribution (Figure 3c) or CpG content and distance to CpG islands
DNA methylation of individual CpG sites in human

adipose tissue is influenced by exercise
We next investigated if there was a difference in DNA
methylation in any of the 476,753 analyzed individual CpG sites
in adipose tissue in response to exercise. A flowchart of the analysis
process is found in Figure 2. SNPs within the probe were not a
criterion for exclusion in this analysis, as the participants are their
own controls, thereby excluding genetic variation within the tested
pairs. Applying FDR correction (q,0.05) resulted in 17,975 CpG
sites, corresponding to 7,663 unique genes, that exhibit differential
DNA methylation in adipose tissue after exercise. Among these
17,975 individual sites, 16,470 increased and 1,505 decreased the
level of DNA methylation in response to exercise, with absolute
changes in DNA methylation ranging from 0.210.9% (Figure 3a
b). Aiming for biological relevance, we further filtered our results
requiring the average change in DNA methylation (b-value) for
each CpG site to be $5% before vs. after exercise. Adding the
criteria with a $5% change in DNA methylation resulted in 1,009
significant individual CpG sites: 911 with increased and 98 with
decreased levels of DNA methylation in response to the six months
exercise intervention. Of those, 723 sites are annotated to one or
more genes, and correspond to 641 unique gene IDs. A
comparison of our 1,009 significant CpG sites with Infinium
probes reported to cross-react to alternative genomic locations
found to decrease in the level of DNA methylation with a

concomitant increase in mRNA expression. Table S3 shows all
significant results of DNA methylation sites with an inverse relation to
mRNA expression in human adipose tissue before vs. after exercise.
Table 1. Clinical characteristics of study participants (n = 23)

with DNA methylation data both before (baseline) and after
the exercise intervention.
Baseline
Age (years)
37.364.4
Weight (kg)
91.8611.0
90.8611.6
0.18
After exercise
DNA methylation in vitro decreases reporter gene

expression
p-value
Characteristics
BMI (kg/m )
28.262.9
27.963.1
0.18
Waist circumference (cm)
97.768.6
95.768.7
0.02
Waist/hip ratio
0.9360.05
0.9260.06
0.01
Fatmass (%)
22.866.0
23.166.6
0.59
Fasting glucose (mmol/L)
5.0160.64
4.9560.59
0.51
2 h OGTT glucose (mmol/L) 6.1761.02
5.8661.47
0.32
HbA1c (%)
4.3160.31
4.3160.34
1.00
Fasting insulin (mU/mL)
6.6062.41
6.8062.86
0.63
VO2max (mL/kg/min)
33.164.6
36.266.2
0.003
Systolic BP (mmHg)
132.5610.2
129.9611.8
0.34
Diastolic BP (mmHg)
79.369.3
74.8610.7
0.04
Pulse (beats/min)
73.9610.6
67.3611.2
0.03
Total cholesterol (mmol/L)
4.9960.71
4.6361.12
0.07
Triglycerides (mmol/L)
1.6361.30
1.2660.98
0.20
LDL (mmol/L)
3.3660.63
3.2460.63
0.41
HDL (mmol/L)
1.0460.21
1.1160.21
0.02
LDL/HDL
3.3160.89
3.0260.92
0.053
RALBP1 belongs to the genes that exhibit increased DNA

methylation in the promoter region in parallel with decreased
mRNA expression in adipose tissue in response to exercise (Figure 4a
b and Table S3). It has previously been shown to play a central role in
the pathogenesis of metabolic syndrome [31] and to be involved in
insulin-stimulated Glut4 trafficking [32]. We proceeded to functionally test if increased DNA methylation of the promoter of RALBP1
may cause decreased gene expression using a reporter gene construct
in which 1500 bp of DNA of the human RALBP1 promoter was
inserted into a luciferase expression plasmid that completely lacks
CpG dinucleotides. The reporter construct could thereby be used to
study the effect of promoter DNA methylation on the transcriptional
activity. The construct was methylated using two different methyltransferases; SssI and HhaI, which methylate all CpG sites or only the
internal cytosine residue in a GCGC sequence, respectively.
Increased DNA methylation of the RALBP1 promoter, as
measured by luciferase activity, suppressed the transcriptional
activity of the promoter (p = 0.028, Figure 4c). When the RALBP1
reporter construct was methylated in vitro using SssI (CG, 94 CpG
sites), the transcriptional activity was almost completely disrupted
(1.460.5), whereas the HhaI enzyme (GCGC, methylating 14
CpG sites) suppressed the transcriptional activity to a lesser extent
(23.4611.6), compared with the transcriptional activity of the
mock-methylated control construct (448.26201.7; Figure 4c).
Data are expressed as mean 6 SD, based on paired t-tests and two-tailed pvalues. BP, blood pressure; LDL, low density lipoprotein; HDL, high density
lipoprotein.
doi:10.1371/journal.pgen.1003572.t001
DNA methylation of obesity and type 2 diabetes

candidate genes in human adipose tissue
(Figure 3d). We found that the CpG sites with altered level of DNA
methylation in response to exercise were enriched within the gene
body and in intergenic regions, while the proximal promoter, in
particular TSS200 and the 1st exon, had a low proportion of
differentially methylated CpG sites (p = 7610220; Figure 3c). In
relation to CpG content and distance to CpG islands, the region
with the highest proportion of significant CpG sites compared to the
distribution on the array was in the open sea, i.e., regions more
distant from a CpG island than 4000 bp. In contrast, the number of
significant CpG sites found within the CpG islands was only half of
what would be expected (p = 2610231; Figure 3d).
We proceeded to investigate if candidate genes for obesity or

T2D, identified using genome-wide association studies [3], are
found among the genes exhibiting changed levels of DNA
methylation in adipose tissue in response to six months exercise.
Among all 476,753 CpG sites analyzed on the Infinium HumanMethylation450 BeadChip and passing QC, 1,351 sites mapped to
53 genes suggested to contribute to obesity in the review by
McCarthy, and 1,315 sites mapped to 39 genes suggested to
contribute to T2D [3]. We found 24 CpG sites located within 18 of
the candidate genes for obesity with a difference in DNA
methylation in adipose tissue in response to the exercise intervention
(q,0.05, Table 4). Additionally, two of those genes (CPEB4 and
SDCCAG8) showed concurrent inverse change in mRNA expression
after exercise (q,0.05). Among the T2D candidate genes, 45 CpG
sites in 21 different genes were differentially methylated (q,0.05) in
adipose tissue before vs. after exercise (Table 5). Of note, 10 of these
CpG sites mapped to KCNQ1 and 6 sites mapped to TCF7L2. A
simultaneous change in mRNA expression was seen for four of the
T2D candidate genes (HHEX, IGF2BP2, JAZF1 and TCF7L2)
where mRNA expression decreased while DNA methylation
increased in response to exercise (q,0.05, Table 5).
Exercise induces overlapping changes in DNA

methylation and mRNA expression
An increased level of DNA methylation has previously been
associated with transcription repression [14]. We therefore related
changes in adipose tissue DNA methylation of individual CpG-sites
(q,0.05 and difference in mean b-values $5%) with changes in
mRNA expression of the same gene (q,0.05) in response to exercise
(Figure 2). We identified 236 CpG sites in 197 individual gene regions
that exhibit differential DNA methylation together with a significant
change in adipose tissue mRNA expression of the corresponding gene
after exercise. Of these, 143 CpG sites (61%) connected to 115 genes
showed an inverse relation to mRNA expression. After exercise, 139
CpG sites showed an increase in DNA methylation and a
corresponding decrease in mRNA expression, including a gene for
one of the GABA receptors (GABBR1), several genes encoding histone
modifying enzymes (EHMT1, EHMT2 and HDAC4) and a
transcriptional co-repressor (NCOR2). Only four CpG sites were
Silencing of Hdac4 and Ncor2 in 3T3-L1 adipocytes is

associated with increased lipogenesis
To further understand if the genes that exhibit differential DNA
methylation and mRNA expression in adipose tissue in vivo affect
adipocyte metabolism, we silenced the expression of selected genes
in 3T3-L1 adipocytes using siRNA and studied its effect on
lipogenesis. Two of the genes where we found increased DNA
4
sample analyzed at four different occasions. Technical reproducibility was observed between all samples, with Pearsons correlation coefficients .0.99 (p,2.2610216, Figure S1a). Secondly, we
re-analyzed DNA methylation of four CpG sites using Pyrosequencing (PyroMark Q96ID, Qiagen) in adipose tissue of all 23
men both before and after exercise (Table S4). We observed a
significant correlation between the two methods for each CpG site
(p,0.05; Figure S1b), and combining all data points gives a
correlation factor of 0.77 between the two methods (p,0.0001;
Figure S1c).
Discussion
This study highlights the dynamic feature of DNA methylation,
described using a genome-wide analysis in human adipose tissue
before and after exercise. We show a general global increase in
adipose tissue DNA methylation in response to 6 months exercise,
but also changes on the level of individual CpG sites, with
significant absolute differences ranging from 0.210.9%. This
data, generated using human adipose tissue biopsies, demonstrate
an important role for epigenetic changes in human metabolic
processes. Additionally, this study provides a first reference for the
DNA methylome in adipose tissue from healthy, middle aged men.
Changes in DNA methylation have been suggested to be a
biological mechanism behind the beneficial effects of physical
activity [18,36]. In line with this theory, a nominal association
between physical activity level and global LINE-1 methylation in
leukocytes was recently reported [37]. More important from a
metabolic point-of-view, a study investigating the impact of long
term exercise intervention on genome-wide DNA methylation in
human skeletal muscle was recently published, and showed
epigenetic alterations of genes important for T2D pathogenesis
and muscle physiology [23]. This relationship between exercise
and altered DNA methylation is here expanded to include human
adipose tissue, as our data show 17,975 individual CpG sites that
exhibit differential DNA methylation in adipose tissue after an
exercise intervention, corresponding to 7,663 unique genes
throughout the genome. Genome-wide association studies have
identified multiple SNPs strongly associated with disease, but still
the effect sizes of the common variants influencing for example risk
of T2D are modest and in total only explain a small proportion of
the predisposition. Importantly, although each variant only
contributes with a small risk, these findings have led to improved
understanding of the biological basis of disease [3]. Similarly, the
absolute changes in DNA methylation observed in response to the
exercise intervention are modest, but the large number of affected
sites may in combination potentially contribute to a physiological
response. Moreover, if the exercise induced differences in DNA
methylation is expressed as fold-change instead of absolute
differences, we observe changes ranging from 6 to 38%.
In regard to the distribution of analyzed CpG sites, most of the
differentially methylated sites were found within the gene bodies
and in intergenic regions, and fewer than expected was found in
the promoter regions and CpG islands. This is in agreement with
previous studies showing that differential DNA methylation is
often found in regions other than CpG islands. For example, it was
shown that tissue-specific differentially methylated regions in the
59UTR are strongly underrepresented within CpG islands [38]
and that most tissue-specific DNA methylation occurs at CpG
island shores rather than the within CpG islands, and also in
regions more distant than 2 kb from CpG islands [39]. It has
further been proposed that non-CpG island DNA methylation is
more dynamic than methylation within CpG islands [40]. The
importance of differential DNA methylation within gene bodies is
Figure 2. Analysis flowchart.

methylation in parallel with decreased mRNA expression in

human adipose tissue in response to exercise (Figure 5ad and
Table S3) were selected for functional studies in a 3T3-L1
adipocyte cell line. HDAC4 was further a strong candidate due to
multiple affected CpG sites within the gene, and both HDAC4 and
NCOR2 are biologically interesting candidates in adipose tissue and
the pathogenesis of obesity and type 2 diabetes [3335]. Silencing
of Hdac4 and Ncor2 in the 3T3-L1 adipocytes resulted in 74%
reduction in the Hdac4 protein level (1.0060.50 vs. 0.2660.20,
p = 0.043; Figure 5e) while the Ncor2 mRNA level was reduced by
56% (1.0060.19 vs. 0.4460.08, p = 0.043; Figure 5f) of control
after transfection with siRNA for 72 hours and 24 h, respectively.
Lipogenesis was nominally increased in the basal state (1.0060.26
vs. 1.4460.42, p = 0.079) and significantly increased in response to
0.1 nM insulin (1.1660.30 vs. 1.5260.34, p = 0.043) in 3T3-L1
adipocytes with decreased Hdac4 levels (Figure 5g). Decreased
Ncor2 levels also resulted in increased lipogenesis in the basal
(1.0060.19 vs. 1.1960.19, p = 0.043) and insulin stimulated
(1 nM; 1.3860.17 vs. 1.7360.32, p = 0.043) state (Figure 5h).
Technical validation of Infinium HumanMethylation450

BeadChip DNA methylation data
To technically validate the DNA methylation data from the
Infinium HumanMethylation450 BeadChips, we compared the
genome-wide DNA methylation data from one adipose tissue
Figure 3. DNA methylation of individual CpG sites. The absolute change in DNA methylation of individual CpG sites with a significant
difference after exercise compared with baseline (q,0.05) ranges from 0.210.9% (A and B). A) Number of sites with increased methylation in adipose
tissue in response to exercise (n = 16,470). B) Number of sites with decreased DNA methylation in adipose tissue in response to exercise (n = 1,505).
Panels C and D show the distribution of CpG sites with a significant change (q,0.05) and an absolute difference $5% in DNA methylation in adipose
tissue before versus after exercise, in comparison to all analyzed sites on the Infinium HumanMethylation450 BeadChip. C) Distribution of significant
CpG sites vs. all analyzed sites in relation to nearest gene regions. D) Distribution of significant CpG sites vs. all analyzed sites in relation to CpG island
regions. *The overall distribution of significant CpG sites compared with all analyzed sites on the Infinium HumanMethylation450 BeadChip was
analyzed using a chi2 test.
DLK1 as markers of preadipocytes, PRDM16 and UCP1 as markers

of brown adipocytes; ITGAX, EMR1, ITGAM as markers of
macrophages; TNF and IL6 representing cytokines and finally
CCL2 and CASP7 as markers for inflammation). Although this
result suggests that there is no a major change in the cellular
composition of the adipose tissue studied before compared with
after the exercise intervention, future studies should investigate the
methylome in isolated adipocytes. Additionally, in previous studies
of DNA methylation in human pancreatic islets, the differences
observed in the mixed-cell tissue were also detected in clonal beta
cells exposed to hyperglycemia [20,21], suggesting that in at least
some tissues, the effects are transferable from the relevant cell type
to the tissue of interest for human biology.
The impact of this study is further strengthened by our results
showing altered DNA methylation of genes or loci previously
associated with obesity and T2D. Although there was no
enrichment of differential DNA methylation in those genes
compared to the whole dataset, this result may provide a link to
the mechanisms for how the loci associated with common diseases
exert their functions [18]. 18 obesity and 21 T2D candidate genes
had one or more CpG sites which significantly changed in adipose
tissue DNA methylation after exercise. 10 CpG sites were found to
supported by multiple studies showing a positive correlation

between gene body methylation and active transcription [40], and
that DNA methylation may regulate exon splicing [41,42]. In this
study, the exercise intervention associated with a decrease in waist
circumference and waist-hip ratio, which suggests reduced
abdominal obesity, a phenotype known to be associated with
reduced risk of metabolic diseases [43]. Indeed, increased levels of
DNA methylation were observed after exercise both in the
promoter region and in the gene body of ITPR2, a locus previously
associated with waist-hip ratio [44]. Furthermore, in addition to
increased VO2max, the study participants responded to exercise
with a decrease in diastolic blood pressure and heart rate, and an
improvement in HDL levels, which are some of the different
mechanisms through which exercise is known to reduce the risk for
T2D and cardiovascular disease [43]. Adipose tissue comprises not
only of adipocytes but a mixture of different cell types. To evaluate
if the cellular composition of adipose tissue may change during
exercise, we looked at the mRNA expression for a number of cell
type specific markers before and after the exercise intervention.
None of these showed any difference in adipose tissue mRNA
expression before vs. after exercise (q.0.05; LEP, PNPLA2, FAS,
LIPE and PPARG as markers of adipocytes; SEBPA/B/D and
Table 2. Changes in adipose tissue DNA methylation in response to a 6 months exercise intervention. Most significant CpG sites
(q,0.005) with a difference in DNA methylation $5%.
Location in relation to
DNA Methylation (%)
Probe ID
Chr
Nearest Gene
Gene region
CpG Island
Before
exercise
After
exercise
Difference p-value
cg04090794
HSP90B3P
TSS1500
Open sea
31.465.1
36.664.6
5.2
2.3861027
0.004
cg05091570
NAV1
Body
CpG Island
30.964.1
37.063.5
6.1
4.7761027
0.004
cg01828733
NAV1
TSS200;Body
CpG Island
40.664.1
46.264.3
5.5
1.1961026
0.004
27
0.004
NR5A2
q-value
(,0.005)
cg24553673
Body
S Shore
33.164.7
39.963.8
6.8
2.38610
cg27183818
Intergenic
Open sea
66.764.7
60.764.3
26.0
7.1561027
0.004
cg26091021
Intergenic
N Shelf
38.963.6
45.463.3
6.5
2.3861027
0.004
cg26297203
Intergenic
N Shelf
52.563.2
57.663.0
5.0
1.1961026
0.004
cg14091208
Body
CpG Island
41.464.6
47.364.8
5.9
1.1961026
0.004
cg09217023
Intergenic
Open sea
57.264.0
62.663.2
5.5
2.3861027
0.004
cg09380805
Intergenic
N Shelf
29.064.0
35.763.8
6.6
7.1561027
0.004
cg17103081
GPR125
Body
N Shelf
63.465.2
68.464.9
5.1
1.1961026
0.004
cg15133208
SNCA
59UTR
N Shore
36.564.8
42.464.8
6.0
1.6761026
0.004
26
0.004
CCDC48
cg14348967
Intergenic
Open sea
31.965.2
37.564.4
5.6
1.67610
cg21817858
Intergenic
CpG Island
46.265.2
51.864.4
5.6
2.3861027
0.004
cg20934416
Intergenic
Open sea
76.464.9
81.763.4
5.3
2.3861026
0.005
cg14246190
EHMT2
Body
N Shelf
65.164.3
70.463.4
5.3
2.3861026
0.005
cg20284982
IER3
TSS1500
S Shore
45.365.5
51.263.6
5.9
2.3861026
0.005
cg12586150
SERPINB1
Body
N Shore
51.965.2
58.464.7
6.5
2.3861026
0.005
cg09871057
STX1A
Body
CpG Island
52.363.5
57.463.2
5.1
1.1961026
0.004
cg18550262
Intergenic
Open sea
39.563.4
45.063.2
5.5
2.3861027
0.004
cg00555695
Body
Open sea
40.363.9
45.863.4
5.5
2.3861027
0.004
26
0.005
PVT1
cg13832372
LHX6
Body
S Shore
25.864.5
31.165.4
5.4
2.38610
cg02725718
10
ENKUR
Body
Open sea
65.663.8
70.863.1
5.2
2.3861026
0.005
cg12127706
11
CTTN
Body
Open sea
54.363.9
59.563.7
5.2
1.1961026
0.004
cg02093168
11
HCCA2
Body
Open sea
61.265.8
67.564.2
6.4
1.1961026
0.004
cg22041190
11
PKNOX2
59UTR
S Shore
36.064.5
41.064.1
5.0
1.6761026
0.004
cg12439006
11
Intergenic
Open sea
64.564.2
69.763.2
5.2
2.3861027
0.004
cg19896824
11
Intergenic
Open sea
53.865.4
60.664.1
6.9
2.3861027
0.004
cg21999471
11
Intergenic
Open sea
41.165.3
46.763.6
5.6
2.3861026
0.005
26
0.004
Crossreactive
probes
48
48
47
47
cg26828839
12
ANO2
Body
Open sea
32.565.3
39.765.5
7.1
1.19610
cg13203394
12
ITPR2
Body
Open sea
56.864.4
63.363.2
6.5
4.7761027
0.004
cg26119796
13
RB1
Body
S Shore
57.064.8
62.464.5
5.4
1.6761026
0.004
cg00808648
14
PACS2
TSS1500
N Shore
44.064.1
49.364.1
5.3
4.7761027
0.004
cg22396498
15
CRTC3
Body
Open sea
59.564.5
64.665.1
5.1
1.1961026
0.004
cg07299078
16
KIFC3
Body;59UTR
Open sea
49.664.3
55.964.9
6.4
2.3861027
0.004
48
cg05797594
16
MIR1910;C16orf74
TSS1500;59UTR
Open sea
51.565.1
57.262.9
5.6
2.3861026
0.005
47
cg05516390
16
ZFHX3
59UTR
N Shelf
41.864.4
49.864.4
8.0
1.1961026
0.004
cg06078469
17
MSI2
Body
S Shore
43.563.6
48.864.2
5.4
4.7761027
0.004
27
0.004
cg22386583
17
Body
Open sea
51.263.8
57.063.5
5.8
4.77610
cg11225357
17
RPTOR
Intergenic
Open sea
45.164.1
50.663.9
5.5
1.1961026
0.004
cg20811236
18
Intergenic
N Shore
60.965.3
68.264.9
7.3
4.7761027
0.004
cg21685776
18
Intergenic
S Shore
51.464.4
56.664.8
5.2
7.1561027
0.004
cg21520111
19
TRPM4
Body
CpG Island
53.463.4
59.064.0
5.5
4.7761027
0.004
cg21427956
20
C20orf160
39UTR
S Shore
37.563.8
43.164.3
5.6
1.1961026
0.004
cg08587504
20
LOC647979
TSS1500
S Shore
62.763.4
68.063.0
5.3
2.3861026
0.005
cg10854441
22
MLC1
TSS1500
N Shelf
51.364.9
57.164.3
5.9
1.6761026
0.004
47
Table 2. Cont.
DNA Methylation (%)
Probe ID
Chr
Nearest Gene
Gene region
CpG Island
Before
exercise
After
exercise
Difference p-value
cg04065832
CDX4
1stExon
CpG Island
50.864.4
56.864.6
6.0
2.3861026
0.005
cg19926635
KCND1
39UTR
S Shelf
49.864.4
55.263.9
5.4
1.1961026
0.004
q-value
(,0.005)
Crossreactive
probes
Data are presented as mean 6 SD, based on paired non-parametric test and two-tailed p-values. Cross-reactive probes: Maximum number of bases ($47) matched to
cross-reactive target as reported by Chen et al. [27].
have altered DNA methylation in response to exercise within the

gene body of KCNQ1, a gene encoding a potassium channel and
known to be involved in the pathogenesis of T2D, and also subject
to parental imprinting [45]. Moreover, exercise associated with

changes in DNA methylation of six intragenic CpG sites in
TCF7L2, the T2D candidate gene harbouring a common variant
Table 3. Changes in adipose tissue DNA methylation in response to a 6 months exercise intervention. Significant CpG sites
(q,0.05) with the biggest change in DNA methylation (.8%).
Probe ID
Chr
cg06550177
cg13906823
cg23397147
17
cg24161057
cg26155520
Nearest
Gene
TSTD1
DNA Methylation (%)
Gene
CpG Island
Before
exercise
After exercise
Difference
(.8%)
p-value
q-value
Intergenic
S Shore
29.667.2
40.667.8
10.9
1.6761025
0.008
TSS200
CpG Island
39.2612.5
50.1615.6
10.9
4.0361025
0.011
24
0.028
Intergenic
Open sea
48.1611.0
58.967.5
10.8
4.75610
TSS200
CpG Island
35.9613.5
46.6614.6
10.7
2.1061025
0.009
Intergenic
Open sea
55.667.1
66.066.6
10.4
7.8761026
0.007
cg05874882
Intergenic
N Shore
34.069.1
44.266.7
10.1
6.0361025
0.013
cg00257920
Intergenic
S Shelf
47.569.7
57.567.7
10.0
1.5361024
0.018
cg03878654
16
ZFHX3
59UTR
N Shore
56.666.7
65.966.9
9.3
1.8161024
0.019
cg08360726
19
PLD3
59UTR
CpG Island
29.768.0
38.9611.8
9.2
1.2861023
0.043
cg26682335
17
ABR
Body
Open sea
60.669.4
69.767.0
9.1
2.5361024
0.022
cg01425666
Intergenic
CpG Island
33.366.8
42.365.7
9.0
2.6261025
0.010
24
0.036
TSTD1
cg01750221
12
Intergenic
Open sea
52.367.5
61.166.4
8.8
8.49610
cg05455393
FHL1
TSS1500
N Shore
52.568.4
61.167.2
8.6
1.2861024
0.017
cg22828884
FOXP1
Body
Open sea
62.664.4
71.264.3
8.6
1.6761025
0.008
cg11837417
19
CLDND2
TSS1500
S Shore
65.366.4
73.965.2
8.6
4.0861024
0.027
cg10323490
THNSL2
TSS1500
N Shore
64.168.1
72.666.2
8.5
9.7661024
0.038
cg03934443
10
Intergenic
Open sea
67.4611.8
75.865.6
8.4
9.7661024
0.038
cg01775802
14
RGS6
Body
Open sea
63.2610.1
71.4610.9
8.2
9.7661024
0.038
cg24606240
NUCKS1
TSS1500
S Shore
55.467.9
63.665.9
8.2
7.3861024
0.034
cg23499846
17
KIAA0664
59UTR
S Shore
54.065.9
62.064.3
8.0
1.0361025
0.007
24
0.025
cg21821308
ASAP2
Body
CpG Island
42.068.5
33.865.9
28.1
3.49610
cg19219423
10
PRKG1
Body
Open sea
55.467.7
47.166.8
28.3
1.8161024
0.019
cg03862437
TMEM44
Body
N Shore
46.367.0
38.065.2
28.3
5.9661026
0.006
cg08368520
FOXK1
Body
Open sea
52.967.8
44.568.0
28.4
9.7661024
0.038
cg01275887
FOXK1
Body
Open sea
66.368.5
57.766.6
28.5
7.3861024
0.034
cg06443678
17
Intergenic
Open sea
51.768.2
43.066.7
28.7
2.9861024
0.024
cg02514003
Intergenic
Open sea
70.666.5
61.768.6
28.9
2.5361024
0.022
cg26504110
19
Body
CpG Island
36.968.7
27.465.1
29.5
2.9861024
0.024
LTBP4
Crossreactive
probes
Data are presented as mean 6 SD, based on paired non-parametric test and two-tailed p-values. Cross-reactive probes: Maximum number of bases ($47) matched to
cross-reactive target as reported by Chen et al. [27].
DNA methylation already have made, and are likely to continue to

make, tremendous advances [48]. High coverage data describing
differences in the levels of DNA methylation between certain
human tissues or cell types [38], as well as differences observed
during development [42], have started to emerge. Regardless,
deeper knowledge about the epigenetic architecture and regulation
in human adipose tissue has been missing until now. We found
that the genetic region with the highest average level of DNA
methylation in adipose tissue was the 39UTR, followed by the gene
body and intergenic regions, and those regions also increased the
level of DNA methylation in response to exercise. This
supports the view that the human methylome can dynamically
respond to changes in the environment [14,15]. One explanation
for the low average levels of DNA methylation observed in the
promoter region (TSS1500/200), 59UTR and the first exon, may
be that these regions often overlap with CpG islands, which are
generally known to be unmethylated. Indeed, our results show a
very low level of DNA methylation within the CpG islands, and
how the level then increases with increasing distances to a CpG
island.
It has long been debated if increased DNA methylation
precedes gene silencing, or if it is rather a consequence of altered
gene activity [40]. The luciferase assay experiments from this study
and others [21,23] suggest that DNA methylation may have a
causal role, as increased promoter DNA methylation leads to
reduced transcriptional activity. Here we further related our
findings of altered DNA methylation to mRNA expression, and we
identified 197 genes where both DNA methylation and mRNA
expression significantly changed in adipose tissue after exercise. Of
these, 115 genes (58%) showed an inverse relation, 97% showing
an increase in the level of DNA methylation and a decrease in
mRNA expression. It should be noted that epigenetic processes are
likely to influence more aspects of gene expression, including
accessibility of the gene, posttranscriptional RNA processing and
stability, splicing and also translation [49]. For example, DNA
methylation within the gene body has previously been linked to
active gene transcription, suggestively by improving transcription
efficiency [42].
Two genes, HDAC4 and NCOR2, with biological relevance in
adipose tissue metabolism were selected for functional validation.
HDAC4 is a histone deacetylase regulated by phosphorylation,
and known to repress GLUT4 transcription in adipocytes [35]. In
skeletal muscle, HDAC4 has been found to be exported from the
nucleus during exercise, suggesting that removal of the transcriptional repressive function could be a mechanism for exercise
adaptation [50]. For HDAC4, we observed increased levels of
DNA methylation and a simultaneous decrease in mRNA
expression in adipose tissue in response to the exercise intervention. Additionally, the functional experiments in cultured adipocytes suggested increased lipogenesis when Hdac4 expression was
reduced. This could be an indicator of reduced repressive activity
on GLUT4, leading to an increase in adipocyte glucose uptake
and subsequent incorporation of glucose into triglycerides in the
process of lipogenesis. NCOR2 also exhibited increased levels of
DNA methylation and a simultaneous decrease in mRNA
expression in adipose tissue in response to the exercise intervention, and furthermore we observed increased lipogenesis when
Ncor2 expression was down regulated in the 3T3-L1 cell line.
NCOR2 is a nuclear co-repressor, involved in the regulation of
genes important for adipogenesis and lipid metabolism, and with
the ability to recruit different histone deacetylase enzymes,
including HDAC4 [51]. These results may be of clinical
importance, since HDAC inhibitors have been suggested in the
treatment of obesity and T2D [18,52].
Figure 4. DNA methylation of RALBP1 is associated with a

decrease in gene expression. A CpG site in the promoter region of
RALBP1 showed A) increased DNA methylation in response to exercise
as well as B) a decrease in mRNA expression. C) In vitro DNA methylation
of the RALBP1 promoter decreased gene expression, as measured by
luciferase activity. The result represents the mean of three independent
experiments, and the values in each experiment are the mean of five
replicates (background control subtracted). Data is presented as mean
6 SEM.
with the greatest described effect on the risk of T2D [3]. This is of
particular interest considering that TCF7L2 is subject to alternative
splicing [46,47] and the fact that gene exons are more highly
methylated than introns, with DNA methylation spikes at splice
junctions, suggesting a possible role for differential DNA
methylation in transcript splicing [42]. In addition to differential
DNA methylation, we also observed an inverse change in adipose
tissue mRNA expression for some of these candidate genes,
including TCF7L2, HHEX, IGF2BP2, JAZF1, CPEB4 and
SDCCAG8 in response to exercise.
The understanding of the human methylome is incomplete
although recently developed methods for genome-wide analysis of
10
TBX15
TMEM160
TUB
ZNF608
ZNF608
cg26104752
cg19694781
cg05003666
cg01610165
cg12817840
Body
62.364.1
15.962.4
76.962.1
Body
Body
TSS200;Body;CpG Island
Body; N Shore
59UTR; S Shore
Body; CpG Island
Body; N Shore
Body
Body; N Shore
59UTR; N Shore
Body; S Shelf
Body
Body
Body
Body; CpG Island
TSS200
TSS200
1stExon
Body
TSS1500; S Shore
21.862.5
10.562.3
21.862.5
56.166.5
5.861.4
63.265.1
40.563.9
84.062.7
87.362.4
60.063.3
76.062.3
67.463.9
79.665.8
76.362.4
9.661.6
31.463.9
39.762.9
40.062.4
56.864.4
56.264.5
1stExon; 59UTR; CpG Island 2.160.4
TSS200; CpG Island
Body
66.564.5
25.863.8
13.162.3
18.462.3
58.367.0
6.861.5
67.864.2
44.263.6
86.362.4
89.561.7
64.163.6
78.662.3
71.962.9
84.863.8
78.262.1
11.162.1
35.263.4
43.563.0
42.762.5
63.363.2
60.164.6
2.660.4
13.761.8
79.261.7
3.9
2.6
23.4
2.3
1.0
4.6
3.7
2.3
2.3
4.0
2.7
4.5
5.2
2.0
1.6
3.9
3.7
2.7
6.5
3.9
0.5
22.2
2.3
4.1
Difference
6610
0.038
0.043
0.043
0.013
561024
761024
661024
161024
0.043
761024
0.013
0.029
361024
9610
0.013
25
0.013
161024
0.013
761025
2610
0.013
161024
25
0.048
861024
0.024
0.043
3610
761024
24
0.013
0.013
161024
461025
0.013
361025
0.013
0.001
561027
9610
0.013
25
0.029
161024
0.039
0.013
0.039
q-value
361024
6610
24
161024
24
p-value
48
48
Crossreactive
probes
171.2622.9
171.2622.9
73.666.7
205.0625.6
374.1637.2
224.1656.3
258.8622.1
182.1620.5
34.464.1
331.3642.3
120.4612.5
205.2623.8
205.2623.8
205.2623.8
146.7634.7
44.2617.0
44.2617.0
44.2617.0
421.5663.1
421.5663.1
65.1611.4
476.3669.3
377.9663.9
218.5646.5
Before
exercise
162.7625.1
162.7625.1
72.768.0
231.1625.7
368.1650.9
214.6644.2
234.9632.9
177.4628.0
34.765.4
331.7639.8
124.0613.5
195.0624.6
195.0624.6
195.0624.6
165.0628.7
57.8641.0
57.8641.0
57.8641.0
401.6691.0
401.6691.0
63.0616.8
448.5658.4
319.3651.1
219.7659.8
After
exercise
mRNA expression
28.5
28.5
20.9
26.1
26.0
29.6
223.9
24.7
0.3
0.4
3.6
210.2
210.2
210.2
18.3
13.6
13.6
13.6
219.9
219.9
22.1
227.8
258.7
1.3
Difference
.0.05
.0.05
.0.05
161023
.0.05
.0.05
161023
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
0.019
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
,161025
.0.05
p-value
0.008
0.008
0.08
0.002
q-value
Data are presented as mean 6 SD, based on paired non-parametric test (DNA methylation) or t-test (mRNA expression) and two-tailed p-values. Cross-reactive probes: Maximum number of bases ($47) matched to cross-reactive
target as reported by Chen et al. [27].
STAB1
MAP2K5
cg20055861
cg08222913
MAP2K5
cg02328326
SDCCAG8
MAP2K5
cg01362115
cg16104450
LYPLAL1
cg16681597
PRKD1
LY86
cg09249494
cg16592301
LY86
cg05021589
NRXN3
LY86
cg02212836
MTIF3
ITPR2
cg13203394
cg16420308
ITPR2
cg07645296
cg20147645
GRB14
cg22380033
MSRA
GPRC5B
cg09141413
cg27519910
ADAMTS9
CPEB4
cg05501868
cg07233933
Nearest Gene
Probe ID
Location
Before
exercise
After
exercise
DNA methylation (%)
Obesity candidate genes
Table 4. Individual CpG sites located within/near candidate genes for obesity [3], with a significant change in DNA methylation in adipose tissue in response to exercise.
HMGA2
IGF2BP2
IGF2BP2
JAZF1
KCNQ1
KCNQ1
KCNQ1
KCNQ1
KCNQ1
KCNQ1
KCNQ1
KCNQ1
KCNQ1
KCNQ1
PRC1
cg06150454
cg13918631
cg02963803
cg01689159
cg03660952
cg04894537
cg06838584
cg08160246
cg13577072
cg15910264
cg19672982
cg24725201
cg25786675
cg04775232
DUSP8
cg01602287
cg17518348
DGKB
cg20836993
HMGA2
CDKN2A
cg07562918
cg17182048
CDKAL1
cg03390300
HMGA2
BCL11A
cg01865786
cg16965605
ARAP1
cg27058763
HHEX
ARAP1
cg15279866
FTO
ARAP1
cg10495997
cg20180364
ARAP1
cg06838038
cg26580413
ARAP1
cg03720898
DUSP8
ADCY5
cg14567877
cg26902557
ADAMTS9
ADAMTS9
cg05501868
cg21527616
11
Body
Body
Body
Body
Body
Body
Body
Body
Body
Body
Body; CpG Island
Body
Body
Body
Body
Body
Body
TSS1500; N Shore
Body
Body
Body; CpG Island
Body
1stExon; CpG Island
Body
Body
Body; S Shelf
59UTR; Body
59UTR; S Shore
Body
Body
Body
Body
Body
82.162.4
66.363.7
91.961.6
70.463.0
81.462.8
67.463.1
60.363.3
46.863.7
40.563.5
51.863.5
80.562.6
59.662.6
66.864.7
54.664.2
78.863.7
81.263.9
70.265.6
46.864.1
61.064.4
49.163.9
75.464.5
68.361.8
16.762.1
85.262.3
64.764.3
57.164.0
56.463.5
61.863.2
42.463.9
73.763.7
80.764.3
63.664.7
62.364.1
Nearest Gene
Probe ID
Location
Before
exercise
84.062.2
62.463.6
93.461.4
73.362.8
84.361.9
71.863.5
63.563.3
44.063.4
44.664.3
55.062.6
83.361.7
62.162.5
70.763.8
58.462.8
83.363.5
84.564.8
75.164.3
50.463.5
64.363.8
52.164.0
79.563.4
70.061.7
18.462.2
87.561.8
67.762.8
60.763.3
58.863.9
64.062.9
46.163.6
77.162.3
84.263.8
67.063.3
66.564.5
After
exercise
DNA methylation (%)
Type 2 diabetes candidate genes
1.9
23.9
1.5
2.9
2.9
4.5
3.2
22.7
4.2
3.2
2.7
2.5
3.9
3.8
4.5
3.4
4.9
3.6
3.3
3.0
4.2
1.7
1.6
2.4
3.0
3.5
2.4
2.2
3.7
3.4
3.5
3.4
4.1
Difference
0.015
261024
0.011
0.042
0.025
0.042
261025
161023
661024
161023
0.015
0.021
0.015
0.013
0.017
261024
361024
261024
961025
361024
0.011
0.011
0.014
0.031
0.011
661025
361025
161024
761024
661025
0.048
0.025
561024
2610
0.015
261024
23
0.021
361024
0.011
0.044
161023
1610
0.037
161023
25
0.015
0.044
261024
1610
23
0.028
0.034
861024
661024
0.041
161023
0.042
0.011
461025
1610
0.013
961025
23
0.015
261024
0.025
0.044
6610
q-value
161023
24
p-value
48
48
48
48
Crossreactive
probes
64.3612.0
67.067.0
67.067.0
67.067.0
67.067.0
67.067.0
67.067.0
67.067.0
67.067.0
67.067.0
67.067.0
238.2626.1
105.6616.5
105.6616.5
32.263.2
32.263.2
32.263.2
172.7624.7
785.0680.7
97.7613.9
97.7613.9
16.863.5
35.965.3
263.1624.7
19.462.1
209.4635.0
209.4635.0
209.4635.0
209.4635.0
209.4635.0
257.2648.3
218.5646.5
218.5646.5
Before
exercise
59.8615.8
66.167.4
66.167.4
66.167.4
66.167.4
66.167.4
66.167.4
66.167.4
66.167.4
66.167.4
66.167.4
218.5628.0
88.4614.9
88.4614.9
34.963.6
34.963.6
34.963.6
144.9630.3
794.4664.7
94.9613.5
94.9613.5
17.863.9
40.567.0
268.3625.1
21.362.7
202.8636.4
202.8636.4
202.8636.4
202.8636.4
202.8636.4
253.1644.8
219.7659.8
219.7659.8
After
exercise
mRNA expression
24.5
20.9
20.9
20.9
20.9
20.9
20.9
20.9
20.9
20.9
20.9
219.7
217.1
217.1
2.7
2.7
2.7
227.8
9.4
22.9
22.9
0.9
4.6
5.2
1.8
26.6
26.6
26.6
26.6
26.6
24.1
1.3
1.3
Difference
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
0.01
,161025
,161025
0.004
0.004
0.004
561025
.0.05
.0.05
.0.05
.0.05
0.023
.0.05
0.009
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
.0.05
p-value
0.047
,0.001
,0.001
0.025
0.025
0.025
,0.001
0.09
0.04
q-value
Table 5. Individual CpG sites located within/near candidate genes for T2D [3], with a significant change in DNA methylation in adipose tissue in response to exercise.
0.008
0.008
0.008
0.008
0.001
0.001
0.001
0.001
.0.05
255.8
255.8
255.8
255.8
6.5
291.7631.4
123.6614.5
189.1617.4
285.2629.9
132.2621.6
187.9622.9
.0.05
291.7631.4
285.2629.9
1.3
474.0674.7
529.9658.9
.0.05
474.0674.7
529.9658.9
0.036
474.0674.7
529.9658.9
28.6
474.0674.7
529.9658.9
6.5
474.0674.7
529.9658.9
0.044
0.042
161023
53.563.2
THADA
THADA
WFS1
ZBED3
cg01649611
cg12277798
cg16417416
cg22051204
59UTR; S Shore
51.163.5
2.4
0.025
161023
66.962.9
TCF7L2
cg23951816
Body
63.963.5
2.9
561024
81.663.3
TCF7L2
cg19226647
Body; S Shelf
77.264.4
4.5
0.015
0.025
261024
5610
42.564.6
4.6
68.463.5
38.565.3
TCF7L2
cg09022607
Body
TCF7L2
cg06403317
Body
63.863.8
4.0
24
0.011
461025
5.561.3
4.461.0
1stExon; N Shore
1.1
0.025
661024
21.263.1
25.564.5
Body; S Shore
24.3
0.037
161023
94.261.7
TCF7L2
cg05923857
Body
92.162.6
2.1
0.034
861024
76.463.8
TCF7L2
cg00831931
Body
72.665.2
3.8
0.015
261024
2.4
84.862.5
82.462.7
PTPRD
cg14545834
Body
71.262.2
PROX1
cg01902845
Data are presented as mean 6 SD, based on paired non-parametric test (DNA methylation) or t-test (mRNA expression) and two-tailed p-values. Cross-reactive probes: Maximum number of bases ($47) matched to cross-reactive
target as reported by Chen et al. [27].
0.008
0.001
255.8
0.13
0.008
.0.05
0.001
255.8
474.0674.7
529.9658.9
.0.05
80.3617.8
1.4
21.267.0
81.8614.5
21.266.2
49
0.015
0.025
261024
77.763.4
73.565.0
68.062.8
Body; CpG Island
4.2
6610
24
Body
3.2
After
exercise
Before
exercise
Nearest Gene
Probe ID
Type 2 diabetes candidate genes
Table 5. Cont.
Location
After
exercise
Before
exercise
DNA methylation (%)
Difference
p-value
q-value
Crossreactive
probes
mRNA expression
Difference
p-value
q-value
In summary, this study provides a detailed map of the human

methylome in adipose tissue, which can be used as a reference for
further studies. We have also found evidence for an association
between differential DNA methylation and mRNA expression in
response to exercise, as well as a connection to genes known to be
involved in the pathogenesis of obesity and T2D. Finally,
functional validation in adipocytes links DNA methylation via
gene expression to altered metabolism, supporting the role of
histone deacetylase enzymes as a potential candidate in clinical
interventions.
Materials and Methods

Ethics statement
Written informed consent was obtained from all participants
and the research protocol was approved by the local human
research ethics committee.
Study participants
This study included a total of 31 men from Malmo, Sweden,
recruited for a six months exercise intervention study, as
previously described [23,53]. Fifteen of the individuals had a
first-degree family history of T2D (FH+), whereas sixteen
individuals had no family history of diabetes (FH2). They were
all sedentary, but healthy, with a mean age of 37.4 years and a
mean BMI of 27.8 kg/m2 at inclusion. All subjects underwent a
physical examination, an oral glucose tolerance test and a
submaximal exercise stress test. Bioimpedance was determined
to estimate fat mass with a BIA 101 Body Impedance Analyzer
(Akern Srl, Pontassieve, Italy). To directly assess the maximal
oxygen uptake (VO2max), an ergometer bicycle (Ergomedic 828E,
Monark, Sweden) was used together with heart rate monitoration
(Polar T61, POLAR, Finland) [53]. FH+ and FH2 men were
group-wise matched for age, BMI and physical fitness (VO2max) at
baseline. Subcutaneous biopsies of adipose tissue from the right
thigh were obtained during the fasting state under local
anaesthesia (1% Lidocaine) using a 6 mm Bergstrom needle (Stille
AB, Sweden) from all participants before and from 23 participants
after the six months exercise intervention (.48 hours after the last
exercise session). The weekly group training program included one
session of 1 hour spinning and two sessions of 1 hour aerobics and
was led by a certified instructor. The participation level was on
average 42.864.5 sessions, which equals to 1.8 sessions/week of
this endurance exercise intervention. The study participants were
requested to not change their diet and daily activity level during
the intervention.
Genome-wide DNA methylation analysis

DNA methylation was analyzed in DNA extracted from adipose
tissue, using the Infinium HumanMethylation450 BeadChip assay
(Illumina, San Diego, CA, USA). This array contains 485,577
probes, which cover 21,231 (99%) RefSeq genes [25,54]. Genomic
DNA (500 ng) from adipose tissue was bisulfite treated using the
EZ DNA methylation kit (Zymo Research, Orange, CA, USA).
Analysis of DNA methylation with the Infinium assay was carried
out on the total amount of bisulfite-converted DNA, with all other
procedures following the standard Infinium HD Assay Methylation Protocol Guide (Part #15019519, Illumina). The BeadChips
images were captured using the Illumina iScan. The raw
methylation score for each probe represented as methylation bvalues was calculated using GenomeStudio Methylation module
software (b = intensity of the Methylated allele (M)/intensity of the
Unmethylated allele (U)+intensity of the Methylated allele
(M)+100). All included samples showed a high quality bisulfite
12
13
Figure 5. Silencing of Hdac4 and Ncor2 in 3T3-L1 adipocytes results in increased lipogenesis. CpG sites in the promoter region of A)
HDAC4 and B) NCOR2 showed increased DNA methylation in response to exercise as well as decreased mRNA expression (CD). Knock-downs were
verified either by E) Western blot analysis (for Hdac4) or F) by qRT-PCR (for Ncor2). Lipogenesis increased in 3T3-L1 adipocytes where G) Hdac4 (n = 5)
or H) Ncor2 (n = 5) had been silenced. Data is presented as mean 6 SEM.
conversion efficiency (intensity signal .4000) [55], and also passed

all GenomeStudio quality control steps based on built in control
probes for staining, hybridization, extension and specificity.
Individual probes were then filtered based on Illumina detection
p-value and all CpG sites with a mean p,0.01 were considered
detected and used for subsequent analysis. In total we obtained
DNA methylation data for 476,753 CpG sites from adipose tissue
of 31 men before and 23 men after the exercise intervention.
Before further analysis, the DNA methylation data was exported
from GenomeStudio and subsequently analyzed using Bioconductor [56] and the lumi package [57]. b-values were converted to Mvalues (M = log2(b/(1-b))), a more statistically valid method for
conducting differential methylation analysis [58]. Next, data was
background corrected by subtracting the median M-value of the
600 built in negative controls and was further normalized using
quantile normalization. Correction for batch effects within the
methylation array data was performed using COMBAT [59]. For
the calculations of global DNA methylation, quantile normalization was omitted and probes reported to be cross-reactive ($49
bases) or directly affected by a SNP (MAF.5%) were removed
[27]. Due to different performance of Infinium I and Infinium II
assays [25], the results based on average DNA methylation are
calculated and presented separately for each probe type. To
control for technical variability within the experiment, one adipose
tissue sample was included and run on four different occasions
(Figure S1a). As the b-value is easier to interpret biologically, Mvalues were reconverted to b-values when describing the results
and creating the figures.
CA, USA). The results represent the mean of three independent

experiments, and the values in each experiment are the mean of
five replicates. Cells transfected with an empty pCpGL-vector
were used as background control in each experiment.
siRNA transfection of 3T3-L1 adipocytes and lipogenesis

assay
For detailed description of siRNA and lipogenesis experiments
see Methods S1. Briefly, 3T3-L1 fibroblasts were cultured at subconfluence in DMEM containing 10% (v/v) FCS, 100 U/ml
penicillin and 100 mg/ml streptomycin at 37uC and 95% air/5%
CO2. Two-day post-confluent cells were incubated for 72 h in
DMEM supplemented with 0.5 mM IBMX, 10 mg/ml insulin and
1 mM dexamethasone, after which the cells were cultured in
normal growth medium. Seven days post-differentiation, cells were
transfected by electroporation with 2 nmol of each siRNA
sequence/gene (Table S5). 0.2 nmol scrambled siRNA of each
low GC-, medium GC- and high GC-complex were mixed as
control. The cells were replated after transfection and incubated
for 72 hours (siRNA against Hdac4) or 24 hours (siRNA against
Ncor2).
Cells harvested for western blot analysis were solubilized and
homogenized, and 20 mg protein was subjected to SDS-PAGE (4
12% gradient) and subsequent transferred to nitrocellulose
membranes. The primary antibody (rabbit polyclonal anti-hdac4;
ab12172, Abcam, Cambridge, UK) was diluted in 5 ml 5% BSA/
TBST and incubated overnight in 4uC. The secondary antibody
(goat anti-rabbit IgG conjugated to horseradish peroxidase;
ALI4404, BioSource, Life Technologies Ltd, Paisley, UK) was
diluted 1:20,000 in 5% milk/TBST. Protein was detected using
Super Signal and ChemiDoc (BioRad, Hercules, CA, USA).
Quantitative PCR (Q-PCR) analyses were performed in
triplicate on an ABI7900 using Assays on demand with TaqMan
technology (Mm00448796_m1, Applied Biosystems, Carlsbad,
CA, USA). The mRNA expression was normalized to the
expression
of
the
endogenous
control
gene
Hprt
(Mm01545399_m1, Applied Biosystems).
To measure lipogenesis, 10 ml tritium labelled ([3H]) glucose
(Perkin Elmer, Waltham, MA, USA) was added followed by
insulin of different concentrations; 0, 0.1, and 1 nM for Hdac4
siRNA and 0 and 1 nM for Ncor2 siRNA experiments, respectively. All concentrations were tested in duplicates. After 1 hour,
incorporation of [3H] glucose into cellular lipids was measured by
scintillation counting. Lipogenesis is expressed as fold of basal
lipogenesis.
mRNA expression analysis

RNA extracted from the subcutaneous adipose tissue biopsies
was used for a microarray analysis, performed using the GeneChip
Human Gene 1.0 ST whole transcript based array (Affymetrix,
Santa Clara, CA, USA), following the Affymetrix standard
protocol. Basic Affymetrix chip and experimental quality analyses
were performed using the Expression Console Software, and the
robust multi-array average method (RMA) was used for background correction, data normalization and probe summarization
[60].
Luciferase assay
The human promoter fragment containing 1500 bp of DNA
upstream of the transcription start site for RALBP1
(Chr18:94740309475529, GRCh37/hg19) was inserted into a
CpG-free luciferase reporter vector (pCpGL-basic) as previously
described [21]. The construct was methylated using two different
DNA methyltransferases; SssI which methylates all cytosine
residues within the double-stranded dinucleotide recognition
sequence CG, and HhaI which methylates only the internal
cytosine residue in the GCGC sequence (New England Biolabs,
Frankfurt, Germany). INS-1 cells were co-transfected with 100 ng
of the pCpGL-vector without (control) or with any of the three
RALBP1 inserts (no DNA methyltransferase, SssI, HhaI) together
with 2 ng of pRL renilla luciferase control reporter vector as a
control for transfection efficiency (Promega, Madison, WI, USA).
Firefly luciferase activity, as a value of expression, was measured
for each construct and normalized against renilla luciferase activity
using the TD-20/20 luminometer (Turner Designs, Sunnyvale,
DNA methylation analysis using PyroSequencing

PyroSequencing (PyroMark Q96ID, Qiagen, Hilden, Germany)
was used to technically validate data from the genome-wide DNA
methylation analysis. PCR and sequencing primers were either
designed using PyroMark Assay Design 2.0 or ordered as predesigned methylation assays (Qiagen, Table S4), and all procedures were performed according to recommended protocols.
Briefly, 100 ng genomic DNA from adipose tissue of 23
individuals both before and after the exercise intervention was
bisulfite converted using Qiagens EpiTect kit. With one primer
biotinylated at its 59 end, bisulfite-converted DNA was amplified
by PCR using the PyroMark PCR Master Mix kit (Qiagen).
14
Methods S1 Detailed descriptions of small interfering RNA

transfection, mRNA expression analysis, lipogenesis assay and
statistical analysis.
(DOC)
Biotinylated PCR products were immobilized onto streptavidincoated beads (GE Healthcare, Uppsala, Sweden) and DNA strands
were separated using denaturation buffer. After washing and
neutralizing using PyroMark Q96 Vacuum Workstation, the
sequencing primer was annealed to the immobilized strand.
PyroSequencing was performed with the PyroMark Gold Q96
reagents and data were analyzed using the PyroMark Q96 (version
2.5.8) software (Qiagen).
Table S1 Baseline clinical characteristics of individuals with

(FH+) or without (FH2) a family history of type 2 diabetes.
(DOC)
Table S2 Average DNA methylation for regions in relation to
nearest gene or CpG islands, separately for Infinium I and II
assays, respectively.
(DOC)
Statistical analysis
Clinical data is presented as mean 6 SD, and comparisons
based on a t-test and two-tailed p-values. Genome-wide DNA
methylation data from the Infinium HumanMethylation450
BeadChip before vs. after the six month exercise intervention
was analyzed using a paired non-parametric test, whereas a paired
t-test was used to compare the mRNA expression. DNA
methylation and mRNA expression data are expressed as mean
6 SD. To account for multiple testing and reduce the number of
false positives, we applied q-values to measure the false discovery
rate (FDR) on our genome-wide analyses of DNA methylation and
mRNA expression [24]. Luciferase activity was analyzed using the
Friedman test (paired, non-parametric test on dependent samples)
and presented as mean 6 SEM. Data from 3T3-L1 adipocyte
experiments showing protein, mRNA and lipogenesis levels are
presented as mean 6 SEM, and the results are based on Wilcoxon
signed-rank test.
Table S3 CpG sites with a change in DNA methylation (q,0.05

and difference in b$5%) concurrent with an inverse change in
mRNA expression (q,0.05) of the nearest gene, in response to the
exercise intervention study.
(DOC)
Table S4 Assay design for technical validation of DNA
methylation data using PyroSequencing.
(DOC)
Table S5 siRNA assays.
(DOC)
Acknowledgments
Ylva Wessman is acknowledged for skilled technical assistance, Targ
Elgzyri for collection of clinical material and Peter Almgren for advice on
the statistical calculations. We acknowledge SCIBLU (Swegene Center for
Integrative Biology at Lund University) Genomics Facility for help with
DNA methylation and mRNA expression analyses.
Supporting Information
Figure S1 Technical validation. A) Technical replicate of one
adipose tissue DNA sample included in the study, analyzed

using the Infinium HumanMethylation450 BeadChip on four
different occasions. BC) Data obtained from all adipose tissue
samples for four CpG sites, from both the Infinium HumanMethylation450 BeadChip (x axis) and using Pyrosequencing
(y axis).
(TIF)
Author Contributions
Conceived and designed the experiments: TR PV KFE HAJ LG CL.
T MDN. Analyzed the data:
Performed the experiments: TR CD TD EN A
TR PV CD TD EH AHO CL. Contributed reagents/materials/analysis
tools: KFE HAJ LG. Wrote the paper: TR PV CD TD EH AHO EN
MDN HAJ LG CL.
References
12. Ronn T, Poulsen P, Hansson O, Holmkvist J, Almgren P, et al. (2008) Age
influences DNA methylation and gene expression of COX7A1 in human skeletal
muscle. Diabetologia 51: 11591168.
13. Sandovici I, Smith NH, Nitert MD, Ackers-Johnson M, Uribe-Lewis S, et al. (2011)
Maternal diet and aging alter the epigenetic control of a promoter-enhancer
interaction at the Hnf4a gene in rat pancreatic islets. Proc Natl Acad Sci U S A 108:
54495454.
14. Bird A (2002) DNA methylation patterns and epigenetic memory. Genes Dev
16: 621.
15. Feinberg AP, Irizarry RA (2010) Evolution in health and medicine Sackler
colloquium: Stochastic epigenetic variation as a driving force of development,
evolutionary adaptation, and disease. Proc Natl Acad Sci U S A 107 Suppl 1:
17571764.
16. Barres R, Osler ME, Yan J, Rune A, Fritz T, et al. (2009) Non-CpG methylation
of the PGC-1alpha promoter through DNMT3B controls mitochondrial density.
Cell Metab 10: 189198.
17. Ling C, Del Guerra S, Lupi R, Ronn T, Granhall C, et al. (2008) Epigenetic
regulation of PPARGC1A in human type 2 diabetic islets and effect on insulin
secretion. Diabetologia 51: 615622.
18. Ling C, Groop L (2009) Epigenetics: a molecular link between environmental
factors and type 2 diabetes. Diabetes 58: 27182725.
19. Volkmar M, Dedeurwaerder S, Cunha DA, Ndlovu MN, Defrance M, et al.
(2012) DNA methylation profiling identifies epigenetic dysregulation in
pancreatic islets from type 2 diabetic patients. EMBO J 31: 14051426.
20. Yang BT, Dayeh TA, Kirkpatrick CL, Taneera J, Kumar R, et al. (2011) Insulin
promoter DNA methylation correlates negatively with insulin gene expression
and positively with HbA(1c) levels in human pancreatic islets. Diabetologia 54:
360367.
21. Yang BT, Dayeh TA, Volkov PA, Kirkpatrick CL, Malmgren S, et al. (2012)
Increased DNA Methylation and Decreased Expression of PDX-1 in Pancreatic
Islets from Patients with Type 2 Diabetes. Mol Endocrinol 26: 120312.
1. Ng SW, Popkin BM (2012) Time use and physical activity: a shift away from
movement across the globe. Obes Rev 13: 65980.
2. Ronti T, Lupattelli G, Mannarino E (2006) The endocrine function of adipose
tissue: an update. Clin Endocrinol (Oxf) 64: 355365.
3. McCarthy MI (2010) Genomics, type 2 diabetes, and obesity. N Engl J Med 363:
23392350.
4. Almgren P, Lehtovirta M, Isomaa B, Sarelin L, Taskinen MR, et al. (2011)
Heritability and familiality of type 2 diabetes and related quantitative traits in
the Botnia Study. Diabetologia 54: 28112819.
5. Groop L, Forsblom C, Lehtovirta M, Tuomi T, Karanko S, et al. (1996)
Metabolic consequences of a family history of NIDDM (the Botnia study):
evidence for sex-specific parental effects. Diabetes 45: 15851593.
6. Isomaa B, Forsen B, Lahti K, Holmstrom N, Waden J, et al. (2010) A family
history of diabetes is associated with reduced physical fitness in the Prevalence,
Prediction and Prevention of Diabetes (PPP)-Botnia study. Diabetologia 53:
17091713.
7. Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin JM, et al.
(2002) Reduction in the incidence of type 2 diabetes with lifestyle intervention or
metformin. N Engl J Med 346: 393403.
8. Tuomilehto J, Lindstrom J, Eriksson JG, Valle TT, Hamalainen H, et al. (2001)
Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with
impaired glucose tolerance. N Engl J Med 344: 13431350.
9. Gluckman PD, Hanson MA, Buklijas T, Low FM, Beedle AS (2009) Epigenetic
mechanisms that underpin metabolic and cardiovascular diseases. Nat Rev
Endocrinol 5: 401408.
10. Fraga MF, Ballestar E, Paz MF, Ropero S, Setien F, et al. (2005) Epigenetic
differences arise during the lifetime of monozygotic twins. Proc Natl Acad
Sci U S A 102: 1060410609.
11. Ling C, Poulsen P, Simonsson S, Ronn T, Holmkvist J, et al. (2007) Genetic and
epigenetic factors are associated with expression of respiratory chain component
NDUFB6 in human skeletal muscle. J Clin Invest 117: 34273435.
15
41. Dayeh TA, Olsson AH, Volkov P, Almgren P, Ronn T, et al. (2013)
Identification of CpG-SNPs associated with type 2 diabetes and differential
DNA methylation in human pancreatic islets. Diabetologia 56: 103646.
42. Laurent L, Wong E, Li G, Huynh T, Tsirigos A, et al. (2010) Dynamic changes
in the human methylome during differentiation. Genome Res 20: 320331.
43. Slentz CA, Houmard JA, Kraus WE (2009) Exercise, abdominal obesity, skeletal
muscle, and metabolic risk: evidence for a dose response. Obesity (Silver Spring)
17 Suppl 3: S2733.
44. Heid IM, Jackson AU, Randall JC, Winkler TW, Qi L, et al. (2010) Metaanalysis identifies 13 new loci associated with waist-hip ratio and reveals sexual
dimorphism in the genetic basis of fat distribution. Nat Genet 42: 949960.
45. Travers ME, Mackay DJ, Nitert MD, Morris AP, Lindgren CM, et al. (2012)
Insights Into the Molecular Mechanism for Type 2 Diabetes Susceptibility at the
KCNQ1 Locus From Temporal Changes in Imprinting Status in Human Islets.
Diabetes 62: 98792.
46. Kaminska D, Kuulasmaa T, Venesmaa S, Kakela P, Vaittinen M, et al. (2012)
Adipose Tissue TCF7L2 Splicing Is Regulated by Weight Loss and Associates
With Glucose and Fatty Acid Metabolism. Diabetes 61: 28072813.
47. Osmark P, Hansson O, Jonsson A, Ronn T, Groop L, et al. (2009) Unique
splicing pattern of the TCF7L2 gene in human pancreatic islets. Diabetologia
52: 850854.
48. Emes RD, Farrell WE (2012) Make way for the next generation: application
and prospects for genome-wide, epigenome-specific technologies in endocrine
research. J Mol Endocrinol 49: R1927.
49. Gibney ER, Nolan CM (2010) Epigenetics and gene expression. Heredity
(Edinb) 105: 413.
50. McGee SL, Fairlie E, Garnham AP, Hargreaves M (2009) Exercise-induced
histone modifications in human skeletal muscle. J Physiol 587: 59515958.
51. Watson PJ, Fairall L, Schwabe JW (2012) Nuclear hormone receptor corepressors: structure and function. Mol Cell Endocrinol 348: 440449.
52. Galmozzi A, Mitro N, Ferrari A, Gers E, Gilardi F, et al. (2012) Inhibition of
Class I Histone Deacetylases Unveils a Mitochondrial Signature and Enhances
Oxidative Metabolism in Skeletal Muscle and Adipose Tissue. Diabetes 62: 732
42.
53. Elgzyri T, Parikh H, Zhou Y, Nitert MD, Ronn T, et al. (2012) First-Degree
Relatives of Type 2 Diabetic Patients Have Reduced Expression of Genes
Involved in Fatty Acid Metabolism in Skeletal Muscle. J Clin Endocrinol Metab
97: E13327.
54. Dedeurwaerder S, Defrance M, Calonne E, Denis H, Sotiriou C, et al. (2011)
Evaluation of the Infinium Methylation 450K technology. Epigenomics 3: 771
784.
55. Teschendorff AE, Menon U, Gentry-Maharaj A, Ramus SJ, Gayther SA, et al.
(2009) An epigenetic signature in peripheral blood predicts active ovarian
cancer. PLoS One 4: e8274.
56. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, et al. (2004)
Bioconductor: open software development for computational biology and
bioinformatics. Genome Biol 5: R80.
57. Du P, Kibbe WA, Lin SM (2008) lumi: a pipeline for processing Illumina
microarray. Bioinformatics 24: 15471548.
58. Du P, Zhang X, Huang CC, Jafari N, Kibbe WA, et al. (2010) Comparison of
Beta-value and M-value methods for quantifying methylation levels by
microarray analysis. BMC Bioinformatics 11: 587.
59. Johnson WE, Li C, Rabinovic A (2007) Adjusting batch effects in microarray
expression data using empirical Bayes methods. Biostatistics 8: 118127.
60. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, et al. (2003)
Exploration, normalization, and summaries of high density oligonucleotide array
probe level data. Biostatistics 4: 249264.
22. Bouchard L, Rabasa-Lhoret R, Faraj M, Lavoie ME, Mill J, et al. (2010)

Differential epigenomic and transcriptomic responses in subcutaneous adipose
tissue between low and high responders to caloric restriction. Am J Clin Nutr 91:
309320.
23. Nitert MD, Dayeh T, Volkov P, Elgzyri T, Hall E, et al. (2012) Impact of
an Exercise Intervention on DNA Methylation in Skeletal Muscle From
First-Degree Relatives of Patients With Type 2 Diabetes. Diabetes 61:
332232.
24. Storey JD, Tibshirani R (2003) Statistical significance for genomewide studies.
Proc Natl Acad Sci U S A 100: 94409445.
25. Bibikova M, Barnes B, Tsan C, Ho V, Klotzle B, et al. (2011) High density DNA
methylation array with single CpG site resolution. Genomics 98: 288295.
26. Maksimovic J, Gordon L, Oshlack A (2012) SWAN: Subset-quantile within
array normalization for illumina infinium HumanMethylation450 BeadChips.
Genome Biol 13: R44.
27. Chen YA, Lemire M, Choufani S, Butcher DT, Grafodatskaya D, et al. (2013)
Discovery of cross-reactive probes and polymorphic CpGs in the Illumina
Infinium HumanMethylation450 microarray. Epigenetics 8: 2039.
28. Bell JT, Tsai PC, Yang TP, Pidsley R, Nisbet J, et al. (2012) Epigenome-wide
scans identify differentially methylated regions for age and age-related
phenotypes in a healthy ageing population. PLoS Genet 8: e1002629.
29. Bocklandt S, Lin W, Sehl ME, Sanchez FJ, Sinsheimer JS, et al. (2011)
Epigenetic predictor of age. PLoS One 6: e14821.
30. Rakyan VK, Down TA, Maslau S, Andrew T, Yang TP, et al. (2010) Human
aging-associated DNA hypermethylation occurs preferentially at bivalent
chromatin domains. Genome Res 20: 434439.
31. Singhal J, Nagaprashantha L, Vatsyayan R, Awasthi S, Singhal SS (2011)
RLIP76, a glutathione-conjugate transporter, plays a major role in the
pathogenesis of metabolic syndrome. PLoS One 6: e24688.
32. Chen XW, Leto D, Chiang SH, Wang Q, Saltiel AR (2007) Activation of RalA
is required for insulin-stimulated Glut4 trafficking to the plasma membrane via
the exocyst and the motor protein Myo1c. Dev Cell 13: 391404.
33. Fang S, Suh JM, Atkins AR, Hong SH, Leblanc M, et al. (2011) Corepressor
SMRT promotes oxidative phosphorylation in adipose tissue and protects
against diet-induced obesity and insulin resistance. Proc Natl Acad Sci U S A
108: 34123417.
34. Sutanto MM, Ferguson KK, Sakuma H, Ye H, Brady MJ, et al. (2010) The
silencing mediator of retinoid and thyroid hormone receptors (SMRT) regulates
adipose tissue accumulation and adipocyte insulin sensitivity in vivo. J Biol
Chem 285: 1848518495.
35. Weems JC, Griesel BA, Olson AL (2012) Class II histone deacetylases
downregulate GLUT4 transcription in response to increased cAMP signaling
in cultured adipocytes and fasting mice. Diabetes 61: 14041414.
36. Barres R, Yan J, Egan B, Treebak JT, Rasmussen M, et al. (2012) Acute exercise
remodels promoter methylation in human skeletal muscle. Cell Metab 15: 405
411.
37. Zhang FF, Cardarelli R, Carroll J, Zhang S, Fulda KG, et al. (2011) Physical
activity and global genomic DNA methylation in a cancer-free population.
Epigenetics 6: 293299.
38. Eckhardt F, Lewin J, Cortese R, Rakyan VK, Attwood J, et al. (2006) DNA
methylation profiling of human chromosomes 6, 20 and 22. Nat Genet 38:
13781385.
39. Irizarry RA, Ladd-Acosta C, Wen B, Wu Z, Montano C, et al. (2009) The
human colon cancer methylome shows similar hypo- and hypermethylation at
conserved tissue-specific CpG island shores. Nat Genet 41: 178186.
40. Jones PA (2012) Functions of DNA methylation: islands, start sites, gene bodies
and beyond. Nat Rev Genet 13: 484492.
16

Six Month Exercise

Hochgeladen von

Dokumentinformationen

Originaltitel

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

Six Month Exercise

Hochgeladen von

Copyright:

Verfügbare Formate

A Six Months Exercise Intervention Influences the

Genome-wide DNA Methylation Pattern in Human

the disease [46]. Individuals with high risk of developing T2D

June 2013 | Volume 9 | Issue 6 | e1003572

Exercise and Human Adipose Tissue DNA Methylation

476,753 sites. No individual CpG site showed a significant

Clinical outcome and global changes in adipose tissue

lian cells, DNA methylation usually occurs in the context of CG

June 2013 | Volume 9 | Issue 6 | e1003572

Exercise and Human Adipose Tissue DNA Methylation

Infinium II assays have less than three underlying CpGs in the

DNA methylation of individual CpG sites in human

June 2013 | Volume 9 | Issue 6 | e1003572

Exercise and Human Adipose Tissue DNA Methylation

found to decrease in the level of DNA methylation with a

Table 1. Clinical characteristics of study participants (n = 23)

DNA methylation in vitro decreases reporter gene

Waist circumference (cm)

Fasting glucose (mmol/L)

2 h OGTT glucose (mmol/L) 6.1761.02

Fasting insulin (mU/mL)

Total cholesterol (mmol/L)

RALBP1 belongs to the genes that exhibit increased DNA

DNA methylation of obesity and type 2 diabetes

We proceeded to investigate if candidate genes for obesity or

Exercise induces overlapping changes in DNA

Silencing of Hdac4 and Ncor2 in 3T3-L1 adipocytes is

June 2013 | Volume 9 | Issue 6 | e1003572

Exercise and Human Adipose Tissue DNA Methylation

Figure 2. Analysis flowchart.

methylation in parallel with decreased mRNA expression in

Technical validation of Infinium HumanMethylation450

June 2013 | Volume 9 | Issue 6 | e1003572

Exercise and Human Adipose Tissue DNA Methylation

DLK1 as markers of preadipocytes, PRDM16 and UCP1 as markers

supported by multiple studies showing a positive correlation

June 2013 | Volume 9 | Issue 6 | e1003572

Exercise and Human Adipose Tissue DNA Methylation

DNA Methylation (%)

PLOS Genetics | www.plosgenetics.org

June 2013 | Volume 9 | Issue 6 | e1003572

Exercise and Human Adipose Tissue DNA Methylation

DNA Methylation (%)

have altered DNA methylation in response to exercise within the

to parental imprinting [45]. Moreover, exercise associated with

DNA Methylation (%)

PLOS Genetics | www.plosgenetics.org

June 2013 | Volume 9 | Issue 6 | e1003572

Exercise and Human Adipose Tissue DNA Methylation

DNA methylation already have made, and are likely to continue to

Figure 4. DNA methylation of RALBP1 is associated with a

June 2013 | Volume 9 | Issue 6 | e1003572

PLOS Genetics | www.plosgenetics.org

Body; CpG Island

Body; CpG Island

1stExon; 59UTR; CpG Island 2.160.4

TSS200; CpG Island

DNA methylation (%)

Obesity candidate genes

Exercise and Human Adipose Tissue DNA Methylation

June 2013 | Volume 9 | Issue 6 | e1003572

PLOS Genetics | www.plosgenetics.org

Body; CpG Island